The Society for Integrative and Comparative Biology
Molecular Anhydrobiology: Identifying Molecules Implicated in Invertebrate Anhydrobiosis1
1 Institute of Biotechnology, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QT, UK
2 Institute of Bioengineering and Agroecology, Department of Biology, National University of Ireland Maynooth, Maynooth, Co Kildare, Ireland
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SYNOPSIS |
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 SYNOPSIS INTRODUCTIONDEHYDRATION-RESPONSIVE GENESHYDROPHILIC PROTEINS IN...NOVEL BIOINFORMATICS FOR...AAV-LEA-1: A DEHYDRATION...COMPLEX REGULATION OF NEMATODE...LEA PROTEIN PROCESSING IN...CONCLUSIONS AND FUTURE...References |
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Studies in anhydrobiotic plants have defined many genes which are upregulated during desiccation, but comparable studies in invertebrates are at an early stage. To develop a better understanding of invertebrate anhydrobiosis, we have begun to characterise dehydration-inducible genes and their proteins in anhydrobiotic nematodes and bdelloid rotifers; this review emphasises recent findings with a hydrophilic nematode protein. Initial work with the fungivorous nematode Aphelenchus avenae led to the identification of two genes, both of which were markedly induced on slow drying (90–98% relative humidity, 24 hr) and also by osmotic stress, but not by heat or cold or oxidative stresses. The first of these genes encodes a novel protein we have named anhydrin; it is a small, basic polypeptide, with no counterparts in sequence databases, which is predicted to be natively unstructured and highly hydrophilic. The second is a member of the Group 3 LEA protein family; this and other families of LEA proteins are widely described in plants, where they are most commonly associated with the acquisition of desiccation tolerance in maturing seeds. Like anhydrin, the nematode LEA protein, Aav-LEA-1, is highly hydrophilic and a recombinant form has been shown to be unstructured in solution. In vitro functional studies suggest that Aav-LEA-1 is able to stabilise other proteins against desiccation-induced aggregation, which is in keeping with a role of LEA proteins in anhydrobiosis. In vivo, however, Aav-LEA-1 is apparently processed into smaller forms during desiccation. A processing activity was found in protein extracts of dehydrated, but not hydrated, nematodes; these shorter polypeptides are also active anti-aggregants and we hypothesise that processing LEA protein serves to increase the number of active molecules available to the dehydrating animal. Other LEA-like proteins are being identified in nematodes and it seems likely therefore that they will play a major role in the molecular anhydrobiology of invertebrates, as they are thought to do in plants.
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INTRODUCTION |
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SYNOPSISINTRODUCTIONDEHYDRATION-RESPONSIVE GENESHYDROPHILIC PROTEINS IN...NOVEL BIOINFORMATICS FOR...AAV-LEA-1: A DEHYDRATION...COMPLEX REGULATION OF NEMATODE...LEA PROTEIN PROCESSING IN...CONCLUSIONS AND FUTURE...References |
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Anhydrobiosis ("life without water") occurs across all biological kingdoms, including bacteria, fungi, animals and plants. Familiar examples include bakers' yeast (Saccharomyces cerevisiae) and "resurrection" plants such as Craterostigma plantagineum, while most higher plants have desiccation tolerant seeds. Members of three invertebrate taxa, bdelloid rotifers, tardigrades and nematodes, can undergo anhydrobiosis at all stages of the life cycle, while brine shrimps (Artemia spp.), have anhydrobiotic embryonic cysts and the chironomid Polypedilum vanderplanki has anhydrobiotic larvae (Keilin, 1959
; Crowe et al., 1992; Bartels and Salamini, 2001; Clegg, 2001; Watanabe et al., 2004). |
DEHYDRATION-RESPONSIVE GENES |
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The molecular mechanisms governing anhydrobiosis are not fully understood, but considerable emphasis has been placed on the role of non-reducing disaccharides, chiefly trehalose (animals, fungi) and sucrose (plants). Recently, however, it has become clear that such sugars are not sufficient for anhydrobiosis (Hoekstra et al., 2001
) and, indeed, that some anhydrobiotic organisms seem not to use them (Tunnacliffe and Lapinski, 2003). Attention has therefore been turning to the definition of other adaptations required for anhydrobiosis and we have begun to characterise these genes in the nematode Aphelenchus avenae. This species requires a period during which water loss is slow and limited before the animal becomes fully desiccation tolerant, probably to allow time to switch on a critical set of genes, which can be identified by their dehydration-responsive nature. During this preconditioning period, trehalose synthase genes are induced (Goyal et al., 2005a) and trehalose biosynthesis occurs (Madin and Crowe, 1975). Of other upregulated genes identified to date, two encode "hydrophilins" (highly hydrophilic proteins; Garay-Arroyo et al., 2000): a novel protein we have called anhydrin, and a polypeptide, Aav-LEA-1, related to plant Group 3 late embryogenesis abundant (LEA) proteins (Browne et al., 2002; Browne et al., 2004). In quantitative PCR experiments, these genes are upregulated by drying and osmotic stress but not oxidative or temperature stresses (Fig. 1).
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HYDROPHILIC PROTEINS IN NEMATODES |
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Little is known about anhydrin, except that it is predicted to be a small, highly hydrophilic protein with a disordered structure; it is not described in other species. In contrast, many LEA proteins are known, mostly in plants (Bray, 1993
; Cuming, 1999), where they are produced in abundance during seed development, comprising up to 4% of cellular protein (Roberts et al., 1993), and are associated with acquisition of desiccation tolerance. At least four Groups of LEA proteins have been defined by expression pattern and sequence (Wise, 2003; Wise and Tunnacliffe, 2004); Group 3 proteins are characterised by a repeated 11-mer amino acid motif whose consensus among plant proteins is TAE/QAAKE/QKAXE, or is more broadly defined as 
E/QXKE/QKXE/D/Q (where represents a hydrophobic residue) (Dure, 1993; Dure, 2001). The Group 3 LEA protein from A. avenae, Aav-LEA-1, contains several copies of a similar 11-mer motif, although there are several differences, e.g., the first amino acid is usually a lysine, and thus is charged as opposed to polar or hydrophobic. Group 3 LEA proteins are highly hydrophilic and unstructured in solution, but, unusually, become more structured on drying (Goyal et al., 2003; Wolkers et al., 2001).
LEA-like proteins or their genes have been found in non-plant species besides A. avenae (Dure, 2001). In the desiccation-tolerant bacterium Deinococcus radiodurans, inactivation of either of two LEA-like genes results in reduced survival upon desiccation (Battista et al., 2001); in the entomopathogenic nematode Steinernema feltiae, LEA-like protein expression is induced by dehydration (Gal et al., 2003); and bdelloid rotifers contain a desiccation-induced LEA-like protein (Tunnacliffe et al., 2005). Indeed, recent experiments suggest that LEA-like proteins could be widespread in invertebrates (B. McGee and A.T., unpublished data). These discoveries suggests that plants, animals and micro-organisms might use LEA proteins in similar ways to combat water stress. Some effect on stress tolerance seems apparent, since plant LEA proteins confer increased resistance to osmotic or freeze stresses when introduced into yeast, and a barley LEA protein improves tolerance to water deficit in transgenic rice and wheat. Furthermore, in vitro, an algal LEA protein diminished freeze damage of the enzyme lactate dehydrogenase (LDH; references in Wise and Tunnacliffe, 2004); a Citrus dehydrin (Group 2 LEA protein) was found to be about 20% more effective than BSA in protecting malate dehydrogenase (MDH) activity after desiccation and rehydration (Sanchez-Ballesta et al., 2004); and a pea mitochondrial Group 3-like LEA protein partially protected fumarase on evaporative drying (Grelet et al., 2005). Reyes et al. (2005) have extended this approach to a range of hydrophilins from different organisms and shown protection of MDH and LDH. Despite these results, the precise function of LEA proteins is still unclear.
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NOVEL BIOINFORMATICS FOR FUNCTION PREDICTION |
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Wise (2002
; 2003) has developed computational tools for the analysis of polypeptides like the LEA proteins with a degree of repetitiveness—Group 3 LEA proteins have up to 13 11-mer motifs—which are recalcitrant to more conventional bioinformatics. One key method determines profiles of peptide distributions in LEA protein groups and then compares them with profiles in proteins of known function. Each LEA protein group shows a characteristic pattern of matches of peptide profiles; one of the matches, for the subgroup of Group 3 LEA proteins to which Aav-LEA-1 belongs, is with molecular chaperones, including DnaK/ Hsp70 and HtpG. Although the significance of this match with two markedly different families of chaperone is currently unclear, it suggests possible functions for Group 3 LEA proteins which are amenable to experiment. |
AAV-LEA-1: A DEHYDRATION-PROTECTANT MOLECULAR CHAPERONE? |
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The "classical" molecular chaperones, such as the Hsp70 family and the Hsp60 chaperonin complexes, are commonly perceived as "heat shock proteins" (Hsps), being upregulated by heat stress, although they also play a vital role in folding nascent proteins under non-stress conditions (Ellis and Hartl, 2003
). Their function as stress combatants is to potentiate refolding of polypeptide structures which have become partially denatured. Besides such "folding" chaperones, both the eukaryotic and prokaryotic cytoplasm also contain "holding" and "disaggregating" chaperones. Holding chaperones include small Hsps which form large multimeric "windowed" complexes capable of passively stabilising protein species in a partially unfolded state, preventing aggregation until stress has abated and refolding by Hsp70 and Hsp60 teams, or spontaneously, becomes possible (e.g., Haslbeck et al., 1999). Disaggregating chaperones include the Hsp100 family which are thought to disentangle protein aggregates, which then serve as clients for folding chaperones (Ben-Zvi and Goloubinoff, 2001).
The nematode LEA protein, Aav-LEA-1, displays some of the properties of a holding chaperone in that it can prevent aggregation of a stress-sensitive protein like citrate synthase (CS) under certain stress conditions. CS aggregates when exposed to elevated temperature; many heat-shock protein chaperones can prevent this aggregation (Buchner et al., 1998). Aav-LEA-1 does not prevent CS aggregation due to heat stress (Goyal et al., 2005b), but this is perhaps not surprising, since Aav-lea-1 is not induced by heat; more likely, given its expression profile, it has a role as a desiccation protectant. Indeed, our recent data indicate that Aav-LEA-1 is able to prevent aggregation of CS on drying: Figure 2 shows a light scattering assay which detects aggregate formation when CS is subjected to several cycles of drying and rehydration. Addition of Aav-LEA-1 inhibits CS aggregation under these conditions and therefore it fulfils one of the criteria for a dehydration-protectant chaperone (Goyal et al., 2005b). In contrast, bovine serum albumin (BSA) does not protect the enzyme, although BSA is widely used as a stabiliser in other contexts. Most proteins, probably including molecular chaperones, are susceptible to desiccation damage. However, LEA proteins, being largely unstructured in solution, cannot be denatured further by desiccation.
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The apparent function of nematode LEA protein as a dehydration-protectant (or "xeroprotectant") macromolecule is unusual, possibly representing a novel form of chaperone, but it is not unique: a recombinant form of the Group 1 LEA protein from wheat, Em, behaves similarly to Aav-LEA-1 in the above desiccation assay. Both types of LEA protein also prevent aggregate formation of CS due to freeze-thaw stress (Fig. 3). However, it is not the case that every natively unstructured protein shows the same protective function, since
-synuclein, implicated in neurodegenerative diseases, does not protect CS from aggregation in drying and freezing assays (L.J.W and A.T., unpublished data). It will therefore be instructive to test other unfolded proteins as potential anti-aggregants in these assays, including classical molecular chaperones, and also to determine whether other client proteins can be protected in the same way as CS; experiments with LDH suggest that LEA protein function is applicable to other targets (Goyal et al., 2005b).
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To be classified as molecular chaperones, however, the LEA proteins must not only prevent aggregation of clients, they must form transient, non-covalent complexes with them (Ellis, 2004
); assessing protein:protein interactions in the dry state could prove technically difficult, however. LEA proteins might, in fact, simply function as "molecular shields," forming a physical barrier between partially unfolded neighbouring proteins and preventing contact between them. Their unordered, flexible structure is ideal for this purpose. The compact globular structure of BSA, for example, is less well suited to this role; additionally, it will itself begin to unfold and contribute to aggregation when dried, something which cannot occur with the unstructured LEA proteins. |
COMPLEX REGULATION OF NEMATODE LEA PROTEIN |
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The gene encoding Aav-LEA-1 is upregulated by dehydration in A. avenae (Fig. 1), as might be expected if it acts to combat desiccation damage. This is further illustrated by a Northern hybridisation experiment showing a time course of dehydration at 90% relative humidity (RH; Fig. 4A), where Aav-lea-1 mRNA concentrations increase from a very low base level to a maximum at
24 hr after imposition of the stress; the amount of mRNA seems to decline slightly over the next 24 hr, but remains at a high level. In marked contrast, however, a Western blot with Aav-LEA-1-specific antiserum indicates that the 16.5 kDa LEA protein is apparently present in nematodes prior to dehydration and disappears gradually over the 48 hr stress period, being replaced by a smaller protein or group of proteins at around 9 kDa (Fig. 4B). Not all proteins behave in this manner, since Western blotting experiments with antibodies against other proteins show no such dehydration-dependent processing. The inverse correlation between gene expression and level of full length Aav-LEA-1 protein is also manifested on rehydration when mRNA subsides to basal levels, but LEA protein reappears on the Western blot, at least for a few hours, before subsiding (K.G. and A.T., unpublished data).
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These surprising observations are reproducible and seem to be the result of several converging phenomena. The first is the presence of LEA protein in control animals which do not contain (much of) its cognate mRNA. A. avenae is grown according to the method of Evans (1970)
in bottles containing both sterilised wheat grain and a fungus Rhizoctonia solani; the nematodes are fungivorous. The harvesting protocol includes several washing steps and overnight settling through a column of water; animals collected in this way contain very little Aav-lea-1 mRNA. However, during the later stages of culture, possibly reflecting nutrient limitation or overcrowding, nematodes swarm up the sides of the bottle and are probably experiencing stress. If these swarming nematodes are recovered straight from the culture bottle, by scraping from the sides of the vessel, they contain significant quantities of mRNA for the LEA protein. If these animals are soaked in water for as little as 10 min, the level of Aav-lea-1 mRNA markedly decreases. As before, drying nematodes at 90% RH leads to marked elevation of mRNA levels (Fig. 5). It seems therefore that expression of the Aav-lea-1 gene and maintenance of mRNA levels are extremely sensitive to the physiological state of the nematode, and that some expression takes place during growth, presumably reflecting a degree of stress in the culture bottles.
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The presence of LEA protein in the control sample of Fig. 4B is presumably then due to Aav-lea-1 gene expression in A. avenae prior to harvesting, but indicates that the half life of the protein significantly exceeds that of its mRNA. The stability of full length Aav-LEA-1 itself seems to be governed by dehydration since drying at 90% RH results in processing of the LEA protein into smaller forms. The Aav-lea-1 mRNA present in dried animals is presumed to be stable in the dry state and, on rehydration, although the gene is switched off and stored mRNA begins to be degraded, there is probably sufficient mRNA present for a short burst of translation, with the resultant LEA protein once again showing a longer half-life than its message under these conditions.
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LEA PROTEIN PROCESSING IN VITRO |
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SYNOPSISINTRODUCTIONDEHYDRATION-RESPONSIVE GENESHYDROPHILIC PROTEINS IN...NOVEL BIOINFORMATICS FOR...AAV-LEA-1: A DEHYDRATION...COMPLEX REGULATION OF NEMATODE...LEA PROTEIN PROCESSING IN...CONCLUSIONS AND FUTURE...References |
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Perhaps the most interesting of the above observations is the processing of LEA protein. This prompts immediate questions: 1) is processing related to desiccation?; and 2) does processed LEA protein function as an anti-aggregant in the same way as the full length protein? To begin to answer these questions, we have used protein extracts from both hydrated and dried nematodes to treat recombinant Aav-LEA-1 in vitro. Figure 6A shows that an activity, presumably enzymatic, can be observed in protein extracts from dehydrating nematodes, which is able to process recombinant LEA protein into shorter forms resembling those seen in vivo. A much lower level of activity is seen in extracts from hydrated animals, suggesting that the processing activity might be specific to the condition of dehydration. The processed recombinant LEA protein obtained by digestion with dried nematode extract maintains its ability to protect CS from aggregation on drying (Fig. 6B). This leads to the hypothesis that processing LEA protein might be a mechanism for A. avenae to increase the level of anti-aggregant activity present in the drying animal.
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CONCLUSIONS AND FUTURE DIRECTIONS |
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SYNOPSISINTRODUCTIONDEHYDRATION-RESPONSIVE GENESHYDROPHILIC PROTEINS IN...NOVEL BIOINFORMATICS FOR...AAV-LEA-1: A DEHYDRATION...COMPLEX REGULATION OF NEMATODE...LEA PROTEIN PROCESSING IN...CONCLUSIONS AND FUTURE...References |
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The in vitro work with Group 1 and Group 3 LEA proteins from plant and animal sources suggests a possible function for these enigmatic proteins which have puzzled molecular biologists for more than twenty years. Protein aggregation during desiccation is likely to be a major potential hazard for anhydrobiotes; LEA proteins acting as molecular chaperones or molecular shields might play an important role in prevention of this aggregation. It will therefore be of great interest to determine whether an anti-aggregant function for LEA proteins is also observed in vivo, either in anhydrobiotic organisms or in engineered heterologous cells.
The apparently widespread occurrence of LEA and other hydrophilic proteins in desiccation-tolerant or -resistant systems is consistent with there being common mechanisms in diverse organisms for combatting damage due to water loss. Other functions for LEA proteins, besides that of preventing protein aggregation, are also possible and it has been suggested that they might be multi-functional, with one potential role as storage proteins in plants (J. Farrant, personal communication). Some LEA or LEA-like proteins are known to associate with cell membranes and have been shown to prevent liposome leakage on desiccation (Sales et al., 2000). In accordance with this, it has been proposed that folding of LEA proteins on membranes occurs in a manner similar to that proposed for -synuclein, which has a role in vesicle management (Koag et al., 2003; M. Oliver, personal communication). -synuclein is found only in vertebrates and is implicated in neurodegenerative diseases where it can accumulate in fibrillar aggregates in brain tissue. Intiguingly, -synuclein shares a number of characteristics with Aav-LEA-1: the human protein is almost the same size at 140 amino acids; it is acidic, hydrophilic and unstructured in solution; it contains several 11-mer repeat motifs with the potential to form -helix; and it becomes structured under some conditions (Lücking and Brice, 2000; Goedert, 2001). There is also a degree of sequence similarity between the nematode LEA protein Aav-LEA-1 and -synuclein towards their N-terminal ends (Fig. 7). The 14-3-3 proteins, a family of eukaryotic adaptor proteins, have also been noted to show relatedness to the synucleins in this region and to share some functional properties with them (Ostrerova et al., 1999). Further investigations of the structural and functional similarities of LEA proteins and synucleins are underway in several laboratories and the outcome is awaited with great interest.
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In conclusion, despite many remaining questions, it seems that the LEA proteins are slowly yielding their secrets at last. Other adaptations associated with anhydrobiosis are now becoming apparent as more dehydration-responsive genes are identified in A. avenae. These include genes encoding several new LEA-like proteins as well as a number of completely novel proteins (W. Reardon and A.M.B., unpublished data). It is likely therefore that, as in resurrection plants, anhydrobiosis in invertebrates is a complex physiological response, requiring the interplay of several different gene products and metabolites. These adaptations will become increasingly well characterised over the next few years, which promise to be an exciting time for molecular anhydrobiology.
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ACKNOWLEDGMENTS |
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This work was supported by the Leverhulme Trust, the Isaac Newton Trust, the Biotechnology and Biological Sciences Research Council, The Royal Society, Enterprise Ireland and Science Foundation Ireland. A.T. is the Anglian Water Fellow in Biotechnology of Pembroke College, Cambridge.
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FOOTNOTES |
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1 From the Symposium Drying Without Dying: The Comparative Mechanisms and Evolution of Desiccation Tolerance in Animals, Microbes, and Plants presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2005, at San Diego, California.
2 E-mail: at10004{at}biotech.cam.ac.uk
3 Present address: Nutrigenomics Research Group, Dept. of Clinical Medicine, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James Hospital, Dublin 8, Ireland
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References |
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SYNOPSISINTRODUCTIONDEHYDRATION-RESPONSIVE GENESHYDROPHILIC PROTEINS IN...NOVEL BIOINFORMATICS FOR...AAV-LEA-1: A DEHYDRATION...COMPLEX REGULATION OF NEMATODE...LEA PROTEIN PROCESSING IN...CONCLUSIONS AND FUTURE...References |
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